Our goal is to define the mechanisms by which microenvironmental cues in fetal hematopoietic organs dictate hematopoietic stem cell fate choices. We hypothesize that PDGF-B signaling is a major signaling pathway that regulates HSC/progenitor balance in fetal HSC niches. Embryos that lack the PDGF-B gene or its receptor PDGF-R- display abnormalities in their hematopoietic system and exhibit loss of structural integrity of the placental labyrinth, a major hematopoietic organ during development. We will now define whether the lack of PDGF-B signaling compromises the establishment of the HSC pool during fetal hematopoiesis and subjects HSCs to premature differentiation (Aim 1). This will be accomplished by quantifying HSCs and committed progenitors in fetal hematopoietic organs in PDGF-B-/- embryos, and by defining the main cellular source of PDGF-B relevant for hematopoiesis. We will subsequently assess whether PDGF-B signaling is required directly in fetal hematopoietic stem/progenitor cells or indirectly in their niches (Aim 2.) This will be achieved by defining the expression pattern of PDGF-receptors in hematopoietic stem/progenitor subsets in mice and humans throughout ontogeny, and by inducing tissue specific ablation of PDGF-receptors in hematopoietic cells or in their niches. Finally, we will address mechanistically how PDGF-B signaling regulates hematopoiesis (Aim 3). This will be accomplished by investigating the functional consequences of in vitro PDGF-B stimulation on HSC/progenitor fate and by defining the key signaling molecules and transcriptional programs that PDGF-B activates in a hematopoietic context. We have established a unique collection of mouse models and experimental tools that allow dissection of the key components in PDGF-B signaling in fetal hematopoietic niches both in mice and humans. In addition to using established Cre mouse strains for tissue specific gene ablation, we have developed a novel technology for placental trophoblast specific gene targeting by using lentiviral infection at the blastocyst stage. We hope that these studies will uncover a new, important role for this well known pathway in regulating hematopoietic stem cell fates, and begin to define the key niche cells and molecular cues that are utilized for communication between HSCs and their niche during development. Understanding how the behavior of the HSC is influenced by its niche will be essential for the establishment of successful in vitro methods for HSC expansion or generation of functional HSCs from pluripotent cells for therapeutic purposes. PUBLIC HEALTH RELEVANCE: The goal of this project is to investigate how PDGF-B signaling regulates hematopoietic stem/progenitor cell balance in fetal hematopoietic niches. We will use mouse models with tissue specific gene ablation as well as human fetal hematopoietic tissues to assess the significance of PDGF-B signaling pathway as a key regulator and/or a marker in hematopoietic cells and their niche. These studies will begin to unravel unique molecular cues utilized in fetal hematopoietic stem cell niches that may ultimately allow us to improve the methods for culturing hematopoietic stem/progenitor cells for therapeutic purposes.